Analysis of circulating extracellular vesicles (EV) have advanced over the last several years but challenges remain to reproducibly analyze samples in a clinical setting. If more sensitive and reproducible methods could be established, they would have far reaching applications in early cancer detection, measuring patient response to drugs, understanding tumor heterogeneity and discriminating different diseases. The problem is potentiated in pancreatic cancer (PDAC), where early detection methods are lacking, reproducibility of measurements has been a problem and an understanding of vesicle heterogeneity is missing. Furthermore, most existing technologies rely on bulk measurements which are inherently less sensitive since markers of interest are diluted (Yang et al., 2017, Sci Transl Med, 9). The goal of this proposal is to explore a novel single EV analysis (SEA) method (Lee et al, 2018, ACSnano, in press) to shed light on tumor EV (TEV) biomarker composition in PDAC. Specifically, we propose to i) integrate the method with on-chip microfluidics for improved sample handling while adding a MAb-DNA barcoding step for signal amplification and multiplexing (aim 1) and ii) expand and test the approach for point-of-care analyses of TEV in clinical samples (aim 2). The proposed single vesicle method has the potential to transform PDAC cancer research and clinical practice. It will allow us to discover the make-up of TEV in clinical specimen with far reaching applications for early detection (analysis of rare proteins) and treatment t evaluation in different types of cancers.